Modern science of genetic engineering or biotechnology has provided a number of useful techniques for the improvement of crop plants. Many of these improvements are based on the genetic engineering of plants which involves the transfer of genes of diverse origins into the inheritable germ line of crop plants such that those genes can then be readily bred into or among elite lines of those plants for use in modern agriculture. There are a number of techniques available for the introduction of foreign genes into plants, a process known as genetic engineering, or plant transformation. In general, the techniques for delivery of the genes into plants are independent of the transferred genes, and any technique which has been worked out for a plant species may be used to deliver any particular gene of interest into that plant. Because the cultivation techniques for different plants are quite different from each other, and because transformation techniques generally require intense laboratory cultivation of transformed plant tissue, transformation processes are often species specific and the specific transformation protocols which are most efficiently used with one species of plants often do not work as well with other species of plants.
Most plant transformation techniques rely on the introduction of foreign genes into individual cells of tissue of that plant species maintained in a tissue culture. For example, the most common plant transformation technique in dicot plants is based on the ability of the bacterium Agrobacterium tumefaciens to transfer a part of its DNA from the bacterium into the genome of individual dicot plant cells in culture. Typically the foreign gene construction which is transferred into the plant cell includes a gene of interest, which is desired to be introduced into the plant germ line, in tandem with a selectable marker which confers upon the transformed plant cell resistance to a chemical selection agent. In the case of Agrobacterium mediated transformation, the transformation is often performed on callus culture, which is a form of undifferentiated plant cell proliferating in tissue culture. The transformation protocol then typically requires the application of the selection agent to the plant callus tissue which contains some transformed cells therein. The purpose of the selectable marker and the selection agent gene is to separate the cells containing the introduced DNA from those cells which have not been transformed, the separation being performed by killing all or substantially all of the non-transformed cells. Such techniques are widely used for Agrobacterium mediated transformation of a wide variety of dicot species.
For a variety of reasons, these techniques do not work well with all plants, even dicot plant species. For soybean, one early transformation technique was developed which resulted in large numbers of transgenic soybean plants, the technique being based on the approach of particle-mediated gene delivery. In a particle-mediated gene delivery process, the DNA to be inserted into the plant cells is coated onto small carrier particles which are then physically accelerated into tissues of the plant. The carrier particles are very small in relation to the cells of the plant so they actually deliver the genetic material into cells of the plant without killing those cells. Particle-mediated gene delivery can be practiced either on cells of culture or upon actually growing differentiated plant tissue. U.S. Pat. No. 5,015,580 describes some of the first efforts to create transgenic soybean plants through the use of particle-mediated transformation techniques.
The technique developed in the above-identified patent was further refined in the process described in U.S. Pat. No. 5,503,998, describing an improved transformation protocol useful for soybean as well as other plants. In that process, no selection agent is used. The reason that a selectable marker was not useful or used in that process was that the process was based upon the transformation of a few number of cells contained in the growing meristem of an immature embryo of a soybean plant. It was difficult to arrive at a level of application of a selection agent for the then commonly used selectable markers that would permit the growing meristem to still grow, yet still prove toxic to most of the non-transformed cells. Therefore, in the absence of a selectable marker, a different system was utilized for soybean created through the process described in U.S. Pat. No. 5,503,998. That process relied instead on an easily detectable marker, referred to as a screenable marker, which was embodied by the gene beta-glucuronidase or GUS, which could easily be detected by a convenient calorimetric assay. In this process, particle mediated gene delivery was used to introduce a foreign genetic construction into the meristem of an immature embryo which was then induced to directly produce soybean shoots. This process produced large numbers of small shoots created from putatively transformed immature embryos. The majority of the shoots so created were not transgenic. However, the availability of an easily detectable screenable marker, and the relative ease with which large numbers of shoots could be assayed for expression of the selectable marker gene made it practical, and in fact, economic, to simply create large numbers of shoots and look for the rare event in which a transformed shoot was identified. This process enabled the ready creation of large numbers of germ line transgenic plants without the use of a selectable marker.
However, it is always desired to improve the efficiency of any transformation protocol. Thus, the incorporation of a method for improving the efficiency of the method described in the identified US patent would be advantageous. One way to increase the efficiency would be to find a selectable marker that does in fact work with the embryonic transformation system described therein.
A different approach to the transformation of soybean was based on the use of the bacteria Agrobacterium tumefaciens to deliver genes into individual plant cells. U.S. Pat. Nos. 5,416,011 and 5,569,834 describe a soybean transformation system based on Agrobacterium-mediated gene delivery to cotyledonary cells of soybean. This system makes use of an antibiotic selectable marker system, which was made to work with this transformation approach.
One herbicide that has received considerable attention as a candidate for use in the development of genetically engineered plants is N-phosphonomethyl-glycine, or glyphosate. Glyphosate inhibits 3-enoylpyruvylshikimate-5-phosphate synthase (EPSP synthase), an enzyme in the shikimic acid pathway. The shikimic acid pathway is a biosynthetic pathway of plants and bacteria in which aromatic amino acids, plant hormones, and vitamins are produced. EPSP synthase catalyzes the conversion of phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid (EPSP).
Glyphosate is an herbicide that is not known to have any adverse environmental effects and which is degraded naturally in the environment. However, glyphosate is nonspecific, in that it is generally effective in inhibiting the growth of both crop plants and weeds. Therefore, glyphosate cannot be effectively applied as a weed control agent when glyphosate intolerant crop plants are present.
In addition to allowing improved weed control, the introduction of glyphosate resistance markers into soybean plants has facilitated the development of genetically engineered soybean plants having advantageous qualities (e.g., higher yields, pest resistance, enhanced nutritional value, and improved storage qualities). Soybean resistant to glyphosate herbicide are already receiving wide acceptance in the marketplace.
Glyphosate tolerant plants can be obtained by engineering the plants to produce higher levels of EPSP synthase (Shah et al., Science 233:478-481, 1986). U.S. Pat. No. 5,633,435, which is hereby incorporated by reference herein, discloses EPSP synthase variants, each of which has a reduced K.sub.i for glyphosate, a low K.sub.m for PEP, and a high EPSP synthase activity. Transgenic plants comprising an expressible gene encoding a glyphosate-tolerant EPSP synthase were found to have increased tolerance for glyphosate-containing herbicides.
Another means by which glyphosate tolerant plants can be obtained is to introduce into plants a gene encoding a protein involved in glyphosate degradation. U.S. Pat. No. 5,463,175, incorporated by reference herein, discloses a gene that encodes glyphosate oxidoreductase (GOX), an enzyme involved in the degradation of glyphosate. Also disclosed are glyphosate tolerant transgenic plants comprising a heterologous glyphosate oxidoreductase gene. The enzyme glyphosate oxidoreductase catalyzes the cleavage of the C--N bond of glyphosate to yield amino methyl phosphonate (AMPA) and glyoxylate. Under aerobic conditions, oxygen is used as a substrate in the reaction. Under anaerobic conditions, other electron carriers such as phenazine methosulfate and ubiquinone serve as electron acceptors. A variant of glyphosate oxidoreductase having a 10-fold lower K.sub.m for glyphosate than wild type glyphosate oxidoreductase and mutants of this variant generated by site specific mutagenesis were also disclosed.
In order for the use of agriculturally useful transgenic plants produced by particle mediated transformation to be economically feasible, it is necessary to obtain germ line transformation of a plant, so that progeny of the plant will also carry the gene of interest. Obtaining transgenic plants that exhibit transient expression of an introduced gene has now become a relatively routine, straightforward procedure. However, obtaining a germ line transformed plant through particle mediated transformation methods currently known to the art is a rare through regularly occurring event.
U.S. Pat. Nos. 5,633,435 and 5,463,175 disclose glyphosate tolerant soybeans obtained by microparticle injection transformation. Although the method involves the introduction of a selectable marker into a plant or plant cell, the transformation process is a labor intensive cell culture method.
Although the method disclosed in U.S. Pat. No. 5,503,998 affords a reduction in the amount of work that must be undertaken to identify a germ line transformation event, identification of germ line transformants remains a very labor-intensive undertaking, requiring large numbers of shoots to be screened. Using this method, the transformation efficiency (expressed as the percentage of germ line transformants obtained per explant subjected to particle bombardment) is only about 0.05%. Therefore, in order to obtain a single germ line transformed plant, one must subject approximately 2000 explants to particle bombardment and screen about 6000-8000 shoots using the tissue-destructive GUS assay.
What is needed in the art is an efficient method for obtaining germ line transformed transgenic soybean plants using glyphosate selection.